RU2497827C2 - Oligosaccharide ingredient - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: oligosaccharide ingredient, which is intended to increase content of sialic acid in baby formula, contains glycosylated amino acids and peptides of general formula R_nSac_m, where R is an amino acid residue, Sac is a monosaccharide selected from a group comprising N-acetyl-neuraminic acid, N-acetyl galactosamine and galactose n has a value from 1 to 10, under the condition that if n=1, then R is a threonine residue or a serine residue and if n has a value between (2) and (10) the peptide contains at least one threonine or serine residue, m has a value from 2 to 4 and at least 20 mol % of the ingredient is N-acetyl-neuraminic acid, where the oligosaccharide component contains 10-25 mol % N-acetyl galactosamine, 10-25 mol % galactose, 20-50 mol % N-acetyl-neuraminic acid and 15-50 mol % threonine or serine or mixture thereof.

EFFECT: obtaining an oligosaccharide ingredient intended for increasing content of sialic acid in baby formula.

6 cl, 2 ex

Description

FIELD OF THE INVENTION

The present invention relates to a high sialic acid oligosaccharide ingredient, food products containing said oligosaccharide ingredient, and methods for producing said oligosaccharide ingredient.

State of the art

Breast milk is known to contain a large amount of indigestible oligosaccharides. In fact, indigestible oligosaccharides are the third largest solid component (after lactose and lipids) of breast milk and are contained in colostrum at a concentration of 12-15 g / l, and in mature milk at a concentration of 5-8 g / l. Breast milk oligosaccharides are very resistant to enzymatic hydrolysis, which suggests that these oligosaccharides can have vital functions that are not directly related to their energy value.

Over the past two decades, the chemical structure of breast milk oligosaccharides has been studied by NMR and mass spectrometry. Each individual oligosaccharide is based on a combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose and / or N-acetylglucosamine, which are connected in many different ways, which leads to a huge number of different oligosaccharides in breast milk - more so far have been identified 130 such structures. Almost all of them have a lactose fragment at the reducing end, while sialic acid (if present) occupies a terminal position at the non-reducing end.

Of particular interest is the large number of sialylated oligosaccharides in breast milk. Sialic acid is a nine-carbon sugar, which is a vital structural and functional component of the brain gangliosides. It is believed that it plays an important role in the transmission of momentum by nerve cells, the formation of memory and contacts between cells. Studies in young rats have shown that supplementation of sialic acid at an early age increases the sialic acid content in brain gangliosides and improves learning ability in well-fed and poorly fed animals, and that these changes persist in adulthood.

Mother's milk is recommended for all infants. However, in some cases, breastfeeding is not enough or it is unsuccessful for medical reasons, or the mother prefers not to breastfeed. For such cases, mixtures for baby food have been developed. However, cow's milk, which is commonly used as the basis for commercially available infant formula, has a much lower sialylated oligosaccharide content compared to human milk. As the composition of mother's milk and the functions of the individual components become more clear, it becomes apparent that increasing the sialic acid content in infant formula based on cow's milk may be desirable.

There are several well-known sources of sialic acid, including free N-acetylneuraminic acid, sialyllactose, and other sialylated oligosaccharides containing sialic acid gangliosides and caseinoglycomacropeptide protein. Caseinoglycomacropeptide or CGMP is a large carbohydrate-rich hydrophilic protein, which is a C-terminal fragment of k-casein, from which it can be eliminated enzymatically or by treatment with acid. In the application US 2005/0096295 proposed to increase the content of sialic acid in infant formulas by including a new CGMP ingredient having an increased concentration of sialic acid. This ingredient has a sialic acid content above 60 mg / g protein, compared to 40-60 mg / g protein in natural CGMP. It can be obtained either by fractionation using anion chromatography, or by partial hydrolysis, followed by fractionation using anion chromatography.

However, there remains a need for other sialic acid rich ingredients that can be used to enrich infant formulas and other nutritional compositions with this important component.

SUMMARY OF THE INVENTION

The present invention describes an oligosaccharide ingredient containing glycosylated amino acids and proteins of the general formula R _n Sac _m , where R is an amino acid residue, Sac is a monosaccharide selected from the group consisting of N-acetylneuraminic acid, N-acetylgalactosamine and galactose, n is from 1 to 10, provided that if n = 1, then R represents a threonine residue or a serine residue, and if n has a value of 2 to 10, the peptide contains at least one threonine or serine residue, m has a value of 2 up to 4, and p at least 20 mol% of this ingredient is N-acetylneuraminic acid.

The described ingredient is a new food grade ingredient rich in sialic acid, which is suitable for addition to nutritional compositions, such as infant formula, to increase the sialic acid content in the composition.

The present invention also encompasses a method of manufacturing an oligosaccharide ingredient containing glycosylated amino acids and proteins of the general formula R _n Sac _m , where R is an amino acid residue, Sac is a monosaccharide selected from the group consisting of N-acetylneuraminic acid, N-acetylgalactosamine and galactose, n has a value of from 1 to 10, provided that if n = 1, then R represents a threonine residue or a serine residue, and if n has a value of 2 to 10, the peptide contains at least one threonine residue or rina, m has a value from 2 to 4, and at least 20 mol% of this ingredient is N-acetylneuraminic acid, comprising the steps of hydrolyzing caseinoglycomacropeptide using an exoprotease and endoprotease to produce a mixture of free amino acids and proteins with a chain length of 2 to 10 and nanofiltration of the obtained hydrolyzed mixture with retention of a fraction having a molecular weight of 1000-2000 Da.

The present invention also encompasses a food product containing the oligosaccharide ingredient described above. In particular, the food product is a baby food or infant formula, but the food product can be any food or drink consumed by children, infants, or adults. Eating a food product containing such an oligosaccharide ingredient can improve the incorporation of sialic acid into brain gangliosides, and thereby improve the corresponding functions of neurons, such as neurotransmission.

Disclosure of invention

In the present description, the following words are given definitions that must be taken into account when reading and interpreting the description, examples and claims.

"CGMP": caseinoglycomacropeptide, i.e. glycomacropeptide, which is cleaved from bovine k-casein by the action of the rennin enzyme.

"Mixture for baby food": a food product for a balanced diet of infants during the first 1-6 months of their life and for partial nutrition after this period. This term includes mixtures for the initial period of feeding and for the subsequent period of feeding.

"Sialic acid": N-acetylneuraminic acid.

The present invention describes an oligosaccharide ingredient containing glycosylated amino acids and proteins of the general formula R _n SaC _m , where R is an amino acid residue. Sac is a monosaccharide selected from the group consisting of N-acetylneuraminic acid, N-acetylgalactosamine and galactose, n has a value from 1 to 10, provided that if n = 1, then R is a threonine residue or a serine residue, and if n has a value of 2 to 10, the peptide contains at least one threonine or serine residue, m has a value of 2 to 4, and at least 20 mol% of this ingredient is N-acetylneuraminic acid, as well as foods for children or adults containing such an oligosaccharide ingre Dent.

Preferably, n is 1-3, and m is 3 or 4.

The described ingredient contains at least 20 mol.% Sialic acid as part of the saccharide chain linked to the hydroxyl group of threonine or serine. Sialic acid may form part of a chain or may itself be a substituent of a monosaccharide moiety in the chain. So, the oligosaccharide ingredient may contain the following monosaccharides:

Compound Mol% N-acetylgalactosamine (GalNAc) 10-25 Galactose (Gal) g 10-25 N-Acetylneuraminic Acid (NeuAc) 50-20

The oligosaccharide ingredient may contain from 15 to 50 mol% of a mixture of serine and / or threonine.

The oligosaccharide ingredient may contain the following glycosylated amino acids or peptides:

NeuAc-α-2,3-Gal-β-l, 3- (NeuAc-α-2,6 -) - GalNAc-R _n

NeuAc-α-2,3-Gal-β-1,3-GalNAc-R _n

Gal-β-l, 3- (NeuAc-α-2,6 -) - GalNAc-R _n

Gal-β-l, 3-GalNAc-R _n

The oligosaccharide ingredient of the present invention can be obtained by hydrolysis of CGMP using an exoprotease and endoprotease to obtain a mixture of free amino acids and proteins with a chain length of 2 to 10, and nanofiltration of the resulting hydrolyzed mixture with retention of a fraction having a molecular weight of 1000 to 2000 Daltons.

CGMP itself is a cheese by-product in which whole milk is treated with the rennin enzyme to precipitate casein. In this process, CGMP is cleaved from k-casein and remains in solution with whey protein. This product is known as sweet whey. CGMP can be separated from whey protein by any method known in the art. A suitable method is described in patent EP 986312.

Hydrolysis can be carried out using any suitable combination of enzymes. One example of a commercially available enzyme system with exo- and endoprotease activity is a product sold under the Flavourzyme® brand by Novozymes Novo Nordisk.

The product of the hydrolysis process is a mixture of free amino acids, glycosylated threonine and serine, glycosylated peptides containing at least one threonine or serine residue, and non-glycosylated peptides, with the peptides having from 2 to 10 amino acid residues in a chain. The resulting mixture was subjected to nanofiltration to separate and retain a fraction having a molecular weight in the range from 1000 to 2000 Daltons. This fraction constitutes the oligosaccharide ingredient of the present invention.

In a preferred aspect of the present invention, the oligosaccharide ingredient described above is incorporated into a food product. In the context of the present invention, the term "food product" covers any usable product. Thus, it can be a product intended for human consumption, in particular a mixture for feeding infants, a mixture for subsequent feeding, baby food, such as porridge for feeding infants, etc. In particular, the oligosaccharide ingredient of the present invention can be included in infant formula, infant formula 1-3 years old, and milk powder or grain mixes.

The food product can be prepared by any method known in the art, according to the type of product, and the oligosaccharide ingredient of the present invention can be added to the product at a suitable stage in the manufacturing process. For example, a formula for feeding infants can be prepared by mixing in suitable proportions a source of protein, any carbohydrate other than lactose, and a source of fat. Emulsifiers can be added if desired. At this point, you can add vitamins and minerals, but usually they are added later to avoid thermal destruction. Any lipophilic vitamins, emulsifiers, etc. can be dissolved in a source of fats before mixing. Then you can add water, preferably passed through a reverse osmosis unit, to form a liquid mixture.

The resulting liquid mixture can then be thermally treated to reduce bacteria. For example, the liquid mixture can be quickly heated to a temperature in the range of from about 80 ° C to about 110 ° C for a period of time from about 5 seconds to about 5 minutes. This can be done by injecting steam or using a heat exchanger, such as a plate heat exchanger.

Then the liquid mixture can be cooled to a temperature of from about 60 ° C to about 85 ° C, for example, by instant cooling. Then the liquid mixture can be homogenized, for example, in two stages: at a pressure of from about 7 MPa to about 40 MPa in the first stage, and from about 2 MPa to about 14 MPa in the second stage. The homogenized mixture can then be cooled to add heat-sensitive components such as vitamins and minerals. Typically, at this point in time, the pH and solids content of the homogenized mixture traditionally lead to standard values.

The homogenized mixture is transferred to a suitable dryer, such as a spray dryer or a freeze dryer, and pulverized. The resulting powder should have a moisture content of less than about 5 wt.%.

The oligosaccharide ingredient of the present invention can be added directly to the infant formula by dry mixing, or, if the mixture has a liquid consistency, in the mixing step described above. In both cases, it will be understood by a person skilled in the art that the addition of this ingredient entails the simultaneous addition of a certain amount of amine nitrogen, and therefore it may be necessary to adjust the protein content in the infant formula or other products into which the ingredient in question is added. The permitted protein content and amino acid profile of infant formula are specified in legislation, such as European Commission Directive 91/3 21 / EEC of May 14, 1991, and a qualified person skilled in the art can easily adjust the protein content and amino acid profile of infant formula with taking into account the additional amine nitrogen introduced with the ingredient of the present invention, as well as the additional content of threonine and serine.

The final concentration of the oligosaccharide ingredient in the food product for feeding children or infants may be from 0.2 to 4.0 wt.%, Preferably from 0.5 to 2.0 wt.% By weight of solids. However, these values should not be construed as limiting and should be adapted to a specific target audience, for example, based on the weight and age or health status of the child or infant. Preferably, the mixture or food product containing the oligosaccharide ingredient of the present invention is given to the baby at each feeding.

Although it is preferable to introduce the described additive in foods intended specifically for the nutrition of children or infants, a positive effect can be achieved by adding the described additive to foods that do not have a specific target audience, or foods intended for adults. For example, the oligosaccharide ingredient of the present invention can be formulated in food products for patients and food products for the elderly. Such foods may include, but are not limited to, milk, yogurt, cottage cheese, cheese, dairy products, fermented milk based products, ice cream, fermented cereal based products, or dairy based products. In addition to the oligosaccharide ingredient of the present invention, a food product, such as an infant formula, may contain one or more additional oligosaccharides that are added separately.

The present invention will be further illustrated using the examples described below.

Example 1

The following is an example of an infant formula composition containing the oligosaccharide ingredient of the present invention.

Nutrient 100 kcal 1 liter Energy value (kcal) one hundred 670 Protein (g) 1.83 12.3 Fat (g) 5.3 35.7 Linoleic acid (g) 0.79 5.3 α-linolenic acid (mg) 101 675 Lactose (g) 11.2 74.7 Oligosaccharide ingredient (g) 0.15 1,0 Minerals (g) 0.37 2,5 Na (mg) 23 150 K (mg) 89 590 Cl (mg) 64 430 Ca (mg) 62 410 P (mg) 31 210 Mg (mg) 7 fifty Mn (mcg) 8 fifty Se (mcg) 2 13

Vitamin A (mcg RE) 105 700 Vitamin D (mcg) 1,5 10 Vitamin E (mg TE) 0.8 5,4 Vitamin K1 (mcg) 8 54 Vitamin C (mg) 10 67 Vitamin B1 (mg) 0,07 0.47 Vitamin B2 (mg) 0.15 1,0 Niacin (mg) one 6.7 Vitamin B6 (mg) 0,075 0.50 Folic Acid (mcg) 9 60 Pantothenic Acid (mg) 0.45 3 Vitamin B 12 (mcg) 0.3 2 Biotin (mcg) 2.2 fifteen Choline (mg) 10 67 Fe (mg) 1,2 8 I (mcg) fifteen one hundred Cu (mg) 0.06 0.4 Zn (mg) 0.75 5

Example 2

50 g of caseinoglycomacropeptide (Biopure GMP, Davisco, USA) was dissolved at a concentration of 10% (w / v) in 500 ml of deionized water. The resulting solution was heated for 30 minutes at 40 ° C with gentle stirring and recirculation through a laboratory tangential flow filtration system (Millipore, USA) with one Pellicon 1000 Dalton filter cartridge (0.1 m ³ ) at an inlet pressure of 1.2 bar and lack of retentate pressure.

Aminopeptidase from Aspergillus oryzae (Flavourzyme 1000L containing 1000 units of aminopeptidase per gram (LAPU / r), Novozymes, Denmark) was then added in an amount of 3500 LAPU per 50 g of protein. After about an hour, diafiltration was started, increasing the retentate pressure to 3 bar at an inlet pressure of 3 bar. After 5 hours, the filtration was stopped and the retentate was collected. Retentate and 7 filtrate fractions collected during the process were lyophilized. Then determined the solids content (DM) of sialic acid in each fraction. The sialic acid content was determined after mild acid hydrolysis and labeling using the DMB method (1.2-diamino-4,5-methylenedioxybenzene dihydrochloride) and analysis on an HPLC instrument with a Shodex C 18 reverse-phase column and a fluorescent detector using an authentic NeuAc as an external standard. Table 1 shows the mass balance and the content of sialic acid in the starting material, filtrate fractions and retentate.

Table 1 Mass (g) NeuAc (% solids) Exit (%) enrichment Raw material fifty 7.5 one hundred 1.00 Filtrate 1 4.32 0.2 8.64 0,03 Filtrate 2 3,1 0.2 6.2 0.02 Filtrate 3 6.1 0.3 12,2 0.04 Filtrate 4 4.29 1.4 8.58 0.18 Filtrate 5 3.56 0.8 7.12 0.10 Filtrate 6 2,8 0.4 5,6 0.05 Filtrate 7 2.94 0.3 5.88 0.04 Retentate 22.6 14.4 45,2 1.92

The contents of galactose and N-acetylgalactosamine in the retentate were determined after acid hydrolysis using high performance anion exchange chromatography (HPAEC) using a Dionex CarboPac PA1 analytical column and a pulse ammeter detector. Briefly, the retentate was dissolved in water and trifluoroacetic acid was added to a final concentration of 2M. The solution was heated for 3 hours at 100 ° C, dried in a stream of nitrogen and again dissolved in water. The sample was analyzed after filtration through a fine filter with a pore size of 22 μm. Quantification was performed using authentic galactose and galactosamine (N-acetylgalactosamine is converted to galactosamine during acid treatment) as external standards. Threonine was quantified by HPLC after acid hydrolysis in 6 N. HC1.

The final dried retentate product contained 29.6 wt.% (NeuAc) _2-3 -Gal-GalNAc-Thr with a structure of the type NeuAc-α-2,3-Gal-β-l, 3- (NeuAc-α-2,6 -) - GalNAc-threonine / peptide with 30 mol.% Sialic acid, 15 mol.% Galactose, 13 mol.% N-acetylgalactosamine and 42 mol.% Threonine.

Claims (6)

1. An oligosaccharide ingredient designed to increase the content of sialic acid in infant formula, containing glycosylated amino acids and peptides of the general formula R _n Sac _m , where R is an amino acid residue, Sac is a monosaccharide selected from the group consisting of N-acetylneuraminic acid, N β-acetylgalactosamine and galactose, n has a value from 1 to 10, provided that if n = 1, then R is a threonine or serine residue, and if n has a value of 2 to 10, then the peptide contains at least one threonine residue or sir ina, m has a value of from 2 to 4, and N-acetylneuraminic acid comprises at least 20 mol.% of the ingredient, where the oligosaccharide component contains from 10 to 25 mol.% of N-acetylgalactosamine, from 10 to 25 mol.% of galactose, from 20 to 50 mol% of N-acetylneuraminic acid; and 15 to 50 mol% of threonine or serine, or a mixture thereof. 2. The oligosaccharide ingredient according to claim 1, in which n has a value from 1 to 3. 3. The oligosaccharide ingredient according to claim 1, in which m is 2 or 3. 4. A mixture for baby food containing the oligosaccharide ingredient according to any one of claims 1 to 3. 5. The baby food mixture according to claim 4, which contains from 0.2 to 4.0 wt.% Oligosaccharide ingredient. 6. A method of manufacturing an oligosaccharide ingredient according to any one of claims 1 to 3, which includes the step of hydrolyzing the caseinoglycomacropeptide using an exoprotease and endoprotease to produce a mixture of free amino acids and peptides with a chain length of 2 to 10 and a nanofiltration step of the obtained hydrolyzed mixture to select a fraction having molecular weight 1000-2000 Yes.